Technique in blood testing



Sept. 1, 1964 G. w. STEVENSON ETAL 3,147,081

TECHNIQUE IN BLOOD TESTING Filed Feb. 27, 1961 //v VEA/TOES Gso/eas PM STEVENSON,

EEA/EST N. CARLSEN,

EDW/A/ F. ARTHUR 5) THE/E ATTORNEYS HARE/5, /(/ECH, Russsu. & KEEN United States Patent 3,147,081 le Patented Sept. 1, 1964 This invention provides an improved method for laboratory use for evaluating acid-base balance within the human body.

It is common practice to measure blood pH in clinical practice; however, pH measurements alone are inadequate to determine status of the blood pH buffer system, the concentration of which determines the capacity of the blood to withstand pH change. The maintenance of a constant hydrogen ion concentration (a pH of approximately 7.36) in the blood is essential to health. The ability of the blood to maintain this constant hydrogen ion concentration is dependent upon its bufier capacity. The blood buffer capacity is determined by measurement of the amount of total carbon dioxide (CO present in the blood. Total CO in the blood is a much more commonly used clinical determination than blood pH in assessment of acid-base imbalance, although both measurements are informative.

One of the most important buifer systems with regard to pH regulation within the blood is the CO bicarbonate pair. Carbon dioxide is found in the blood as dissolved CO or carbonic acid (H CO and as the bicarbonate radical (HCO Various methods are now available for determining total CO or its equivalent, the sum of concentrations of dissolved CO (H CO and bicarbonate radical. Total CO is normally expressed in millimoles per liter (mM/l.). The sum of the concentrations of dissolved CO and bicarbonate ions expressed in mM/l. represents the amount of CO in the blood.

Two of the more widely used methods for evaluation of acid-base balance are the Van Slyke manometric method which measures the plasma total CO content and the titrimetric technique which determines only the plasma bicarbonate. It will be appreciated that plasma total CO includes plasma bicarbonate and dissolved C0 The titrimetric technique, while being relatively simple and rapid, lacks the accuracy of the manometric method. The Van Slyke manometric method, while providing accurate results generally, does require special equipment and considerable operator skill.

More recently another method has been developed for evaluating the blood buffer capacity. This new method determines the initial pCO of the blood and involves simple pH measurements after equilibration of the blood with known CO gas mixtures. Each blood specimen, depending upon its buifer capacity, will initially exist in equilibrium with a gas mixture of a particular CO content. When a blood specimen is said, for example, to have a pCO value of 47, this means that the particular blood sample exists in equilibrium with a gas mixture having a C0 partial pressure of 47 millimeters of mercury (other units of pressure could, of course, be employed). A blood specimen is in CO equilibrium with a C0 containing gas mixture when no net transfer of CO occurs either way between the blood and the gas mixture. It has been shown that the dissolved CO (H CO content expressed in mM/l. may be determined from the relationship H CO =0.03 pCO the factor 0.03 being a solubility and proportionality factor.

The dissolved CO (carbonic acid or H CO )-bicarbonate bulfer pair exists in equilibrium according to the relationship:

H CO SH++HCOf (1) Let K, be the dissociation constant of Equation 1 and then,

log H =log Kai-log g??? (4) log H ==log K,,llog 2% 5 pH=pK +log M2 (6) From the foregoing Equation 6, which is sometimes known as the Henderson-Hasselbalch equation, it will be seen that if two of three unknown factors (pH, HCO and H CO can be determined, the third can be calculated. The term pK of the Henderson-Hasselbalch equation is a constant having the value 6.10 at 38 C. The pH of blood, of course, is readily determinable by usual electrometric measurements. The dissolved CO or H CO expressed in mM/l. can be readily calculated from the above mentioned relationship H CO =O.03 pCO if the p00 value can be measured. Thus it is seen by determining pH and p00, of a blood specimen, it is possible by employing the Henderson-Hasselbalch equation to determine plasma bicarbonate (the term HCO of the subject equation). The plasma total CO content, which is a measure of blood bufier capacity, is then simply calculated by adding the plasma bicarbonate (HCO3 content to the H CO content, both expressed in mM/ 1. a

In some existing methods for determining the pCO of blood, the blood sample is shaken with a C0 containing gas of known CO content until equilibrium is reached, or the CO containing gas may be bubbled through several milliliters of the blood sample. Bubbling and shaking require about 10-15 minutes to reach equilibrium. The blood specimen after such a treatment will have a pCO value equivalent tothe partial pressure of the CO present in the gas mixture, i.e. a blood equilibrated with a C0 containing gas having a C0 partial pressure of say 47 mm. will have an equilibrated pCO of 47. The pH of the equilibrated blood sample is then measured, requiring a transfer of the blood sample from the bubbling or shaking device to a pH meter. For purposes of illustration, it will be assumed that the equilibrated blood sample has a pH of 7.4 and a p00 value of 42.6. These two values are plotted on the graph of FIG. 1 which has as its abscissa values of pH and as its ordinate values of p00 The blood sample before equilibration had an initial pH value of 7.36. However, it is not possible at this time to plot the initial pH value on the graph for the reason that the initial p00 value of the blood specimen is unknown. In order to determined the initial pCO value, it will be necessary to determine the slope of the line passing through the plot of the equilibrated pH and the equilibrated pCO This is usually achieved in the now existing pCO determinationsby subjecting the blood sample to a second equilibration with a C0 containing gas having a difierent partial pressure of CO than the first equilibrating gas. The blood sample is returned to the shaking or bubbling device and there exposed for an adequate length of time to reach equilibrium with the second gas mixture. It will be assumed that the gas mixture has a C partial pressure of 60, thus giving the equilibrated blood specimen a p00 of 60, and that the second time equilibrated blood specimen has a pH of 7.28. The two values of the second time equilibrated blood specimen are plotted on FIG. 1 as a second point and the line shown is drawn to connect the two plotted points. The initial pH value (7.36) of the bloodbefore equilibration is plotted along the line and from this plot the initial pCO value of the blood specimen is found to be 47.

Knowing the initial pCO value of the blood specimen, the dissolved CO (H CO value expressed in mM/l. can be calculated from the foregoing relationship of H CO =0.03 pCO The dissolved CO (H CO content of the blood specimen is thus found to be 1.4 mM/l. The plasma bicarbonate or HCO content of the blood before equilibration is readily found by substituting the now known values of H CO and pH into the Henderson-Hasselbalch equation [Equation 6]. The plasma bicarbonate content is thus calculated to be 25 mM/l., giving a total CO content of 26.4 mM/l.

The conventional method described above for measuring the pCO of blood and thus determining the blood buffer capacity is time consuming and because of the requirement of transferring the blood specimen between apparatus is subject to some inaccuracy. It is possible to shorten analysis time somewhat by employing only a single equilibrating gas mixture and utilizing a predetermined slope (determined from several average bloods) for plotting of the graph of FIG. 1 but, even with the use of a single equilibration, an analysis will require 20 or more minutes.

It has now been discovered that a blood pCO determination may be simplified and the length of time required for the determination significantly shortened without sacrifice of accuracy. In its broad aspects, the improved process of the invention involves supplying a blood specimen to a sample chamber and there determining the pH of the specimen. Following the pH determination of the blood specimen, a film of the blood is formed within the sample chamber which film is equilibrated with a CO -containing stream of known CO content. Thereafter, the pH of the equilibrated blood film is determined. The improved technique is very rapid, requiring only 15 seconds or so for the equilibration compared with 10 to minutes now required where the cO -containing stream is bubbled through the blood specimen. Additionally, in the practice of the bubbling technique it is necessary to transfer the blood specimen from the pH meter to the bubbling device. Such transfer does not occur in the improved process of the invention. The CO -containing gas stream is preferably saturated with water vapor to avoid extraction of water from the blood of the film, which may interfere with the accuracy of the determination.

In a preferred embodiment of the process of the invention, the sample chamber containing the blood specimen, following the initial pH determination, is swept with a cO -containing gas stream of a known CO content, thus effecting a removal of the bulk of the blood specimen from the chamber, leaving a film of the blood specimen. Flow of the CO -containing gas stream is continued for several seconds to bring about an equilibration of the CO content of the gas stream and blood film. Because of the thinness of the blood film, the equilibration can be effected in a very short time. Thereafter the pH of the blood film is determined. For a most accurate determination the blood film is equilibrated with a second Co -containing gas stream of a known and different CO content than the first gas stream. Following the second equilibration, the blood film is subjected to another pH determination. It is feasible, as explained above in the discussion of the conventional meth- :1: 0d of pCO determination, to employ a single equilibration and use a known slope for plotting of the graph of FIG. 1.

The information gathered from the improved process of the invention is used in the same fashion as described heretofore in the discussion of the conventional method for measurement of blood pCO The apparatus required is relatively simple and essentially the same as that commonly employed for blood pH measurements. The blood specimen used may be the whole blood and where the whole blood is employed, the plasma bicarbonate content is determined and not whole blood bicarbonate which latter determination would require rupture of the blood cells. Plasma bicarbonate content is more commonly determined than Whole blood bicarbonate content in the clinical laboratory. The earlier mentioned Van Slyke manometric technique for plasma and the plasma bicarbonate titrimetric technique require separation of the plasma from the serum of the whole blood, which necessitates a preliminary centrifugation of the blood. The blood specimen employed in the process of our invention may be either whole blood, serum or plasma. In bicarbonate determination of whole blood it would be necessary as mentioned before to rupture the blood cells.

Other advantages and objects of our invention will become more apparent to those skilled in the art from the following description of a preferred form of the invention, it being understood that other modifications and changes may be made. In the drawings:

FIG. 1 is a graph having for its abscissa and ordinate respectively the pH and pCO of a blood specimen handled in accordance with a blood pCO determination with the ordinate having a logarithmic scale;

FIG. 2 is an elevational view partially in section of a blood testing device adapted to the practice of the process of the invention and including a pH assembly including a reference electrode and a test electrode, with the test electrode removed from the assembly;

FIG. 3 is a plan view of the blood testing device of FIG. 1 showing that the reference electrode is placed to one side of the device proper; and

FIG. 4 is a sectional view taken along line 44 of FIG. 2.

A blood testing device 10 formed principally of glass suitable for the practice of the process of the invention as illustrated in FIGS. 1-3 inclusive comprises a cannula 12, a handle 14 formed integrally with the cannula 12, a hollow glass electrode holder 16 opening into a small reservoir or chamber 18 of the cannula 12, and a reference electrode 20 spaced laterally of the cannula 12 and connected through a bridge 22 and a very small hole 24 to the interior of the glass electrode holder 16. The glass electrode holder 16 is shaped to receive a glass electrode assembly 26 providing a ground glass joint. The details of the glass electrode assembly 26 and the reference electrode 20 need not be discussed in detail here as they are conventional and do not constitute a part of the instant invention. The glass electrode assembly 26 has a glass electrode tip 28, made of very thin glass which is responsive to changes of hydrogen ion concentration outside of it. The lower end of the glass electrode assembly 26 includes a tapered male ground section 30 which with positioning of the glass electrode assembly within the glass electrode holder 16 engages the lower interior portion of the holder 16. With the glass electrode assembly 26 positioned within the electrode holder 16, the glass tip 28 assumes the position indicated in dotted lines in FIG. 2 within the small chamber 18 of the cannula 12.

The cannula 12 has a first vertical portion 32 to which the aforementioned handle 14 is attached. The cannula 12 at the lower end of the first vertical portion is curved upwardly forming a short, second vertical portion 34 which houses the aforementioned small chamber 18. Beyond the second vertical portion 34 the cannula 12 has an upwardly sloping segment 36 which at its upper end levels off into a horizontal segment 38. The cannula 12 leading to and from the chamber 18 has a bore 40 of very small diameter. The bore 40 preferably has a diameter in the range of .25 mm. to .75 mm. In the use of the device, the blood specimen is introduced into the top of the first vertical portion 32 of the cannula 12. The blood specimen is discharged through the exit of the horizontal segment 38. In one preferred embodiment, the bridge 22 contains a saturated potassium chloride solution which surrounds a mercurous chloride tube 42. The potassium chloride solution seeps in a small amount through the small hole 24 to provide a continuous electrolyte solution film between the glass electrode holder 16 and the glass electrode assembly 26 (within the ground glass joint).

Purging of the blood specimen from the chamber 18 leaves a blood film over the outside of the glass tip 28 which connects with the electrolyte solution film extending from the hole 24 down to the bottom of the ground joint which constitutes the reference junction. The upper end of the first vertical portion 32 of the cannula 12 is preferably closed with a conventional serum cap (not illustrated) having a rubber diaphragm.

EXAMPLE I The blood testing device 10 is mounted in a waterfilled constant temperature bath (37 C.) and filled with a pH 7.4 bufler solution. After two minutes, the needle of the pH meter is set at 7.4. The buffer is removed from the chamber 18 of the cannula 12 by flushing about mls. of 0.9% sodium chloride solution through it. The sodium chloride solution is expelled by means of an air-filled syringe. The needle of the syringe is inserted through the serum cap of the device which closes the cannula to the atmosphere. Heparinized blood is collected anaerobically in a clean syringe which is shaken gently to mix the blood cells and plasma. The blood is then injected through the serum cap and cannula passage into the chamber 18, filling the chamber and sweeping air bubbles and air exposed blood beyond the chamber 18 and out of the device 10. This requires about 0.5 ml. of blood. After two minutes the pH of the blood is measured (pH The pCO is determined immediately following the pH blood measurements, using the same specimen of blood. About one ml. of water had previously been introduced into a gas bubbler tube equipped with a short flexible hose and syringe needle. The gas bubbler tube is mounted within the constant temperature bath. The water of the gas bubbler tube is equilibrated with a 6% CO gas mixture flowing through it for about a five minute period. With the gas flow rate just fast enough to expel the blood from the chamber 18, the syringe needle connected to the gas bubbler tube is inserted through the serum cap of the device and the blood substantially expelled from the chamber 18, leaving a blood film coating the tip 28 of the glass electrode assembly 26. The pH of the blood film is measured after about fifteen seconds of gas flow.

The blood specimen of the example was found to have a blood pH value of 7.36 and a film pH value of 7.40 upon equilibration of the film with the 6% CO -containing gas. The pCO value of the equilibrated blood film is 42.6 mm. Hg which represents the partial pressure of the CO in the gas stream, taking into account the water vapor partial pressure of 50 mm. With reference to FIG. 1, a point is plotted on the graph to correspond to the film pH value of 7.40 and pCO value of 42.6.

The blood film was equilibrated with a second CO containing gas stream having a C0 content of 8.45% (pCO of the water saturated gas 60 mm. Hg). The blood film after the second equilibration was found to have a pH of 7.28. A second point is plotted on the graph of FIG. 1 to correspond to the pH of 7.28 and pCO of 60 mm. Hg. A line is drawn connecting the two points. The initial pCO value of the blood corre sponding to the original pH, 7.36, is found to be 47 mm.

Hg. Using the relationship H CO =0.03 X p00 the H CO content of the blood in mM/l. is l.4=(0.03 47). Knowing the pH and the H CO content of the original blood, it is possible to employ Equation 6 above, the Henderson-Hasselbalch equation, to determine the plasma bicarbonate. The plasma bicarbonate content is found to be 25 mM/l., giving a total CO content of 25,+ 1.4 or 26.4 mM/l.

Instead of running the second CO gas equilibration of the blood film, it would have been possible to have drawn a line through the point defined by the first equilibration with a predetermined slope characteristic of most bloods. While this latter procedure is not as accurate as may be obtained through a second equilibration, it will for the most purposes give adequately accurate results and shorten the process time somewhat.

EXAMPLE II pH VALUES OF WHOLE OXALATED BLOOD SAMPLES EQUILIBRA'IED WITH 6% CO Filnl Bubbling EXAMPLE III Several blood samples were equilibrated with 6% CO using the bubbling technique and their pH values determined. Similar measurements on plasma separated from the whole blood were found to be nearly identical to the whole blood determinations. The measurements were then repeated using the film technique of the invention and the results are reported in Table II.

Table II pH VALUES FOR "WHOLE BLOOD AND PLASMA SAMPLES EQUILIBRATED -WITH 6% CO1 Whole Plasma Difierence Blood Mean=0. 004

It will be noted that the pH values for whole blood and plasma are nearly identical. This example clearly demonstrates that there is no need to centrifuge the blood to separate the serum or the plasma from the red blood cells in preparation for the analysis as is necessary with the Van Slyke manometric method and the titrimetric technique.

Although an exemplary embodiment of the invention has been disclosed herein for purposes of illustration, it will be understood that various changes, modifications, and substitutions may be incorporated in such embodiment without departing from the spirit of the invention as defined by the following claims which are limited to the method of the invention, the apparatus described herein being covered by our continuing application, Serial No. 372,585, filed June 4, 1964, and assigned to the same assignee as the present application.

We claim:

1. In a blood pCO determination employing pH measurements of a blood specimen, the improvement comprising:

supplying the blood specimen to a sample chamber and there determining the pH of said blood specimen; forming a film of said blood specimen within the sample chamber and equilibrating the blood film with a C containing gas stream of known CO content; and thereafter determining the pH of the equilibrated blood film.

2. A blood pCO determination in accordance with claim 1 wherein the blood film is subsequently equilibrated with a second CO -Containing gas stream of a known but different CO content than the first gas stream and thereafter subjecting the blood film to another pH determination.

3. A blood pCO determination in accordance with claim 1 wherein the Co -containing gas stream is substantially saturated with water vapor.

4. In a blood pCO determination employing pH measurements of a blood specimen, the improvement comprising:

holding the blood specimen in a sample chamber and there determining its pH value;

sweeping the sample chamber with a Co -containing gas stream having a known CO content and substantially saturated with water vapor, thereby removing the bulk of said blood specimen and forming a film of said blood specimen within the sample chamber;

continuing flow of the cO -containing gas stream to effect an equilibration of the CO content of gas stream and blood film; and

thereafter determining the pH of the blood film.

5. A blood pCO determination in accordance with claim 4 wherein the blood film is subsequently equilibrated with a second CO -containing gas stream of a known but dilferent CO content than the first gas stream, said second CO -containing gas stream being substantially saturated with water vapor, and thereafter subjecting the blood film to another pH determination.

6. In a blood pCO determination employing pH measurements of a blood specimen, the improvement comprising:

holding the blood specimen in a sample chamber and there determining its pH value;

sweeping the sample chamber with a CO -cOntaining gas stream having a known CO content, thereby removing the bulk of the blood specimen and forming a film of said blood specimen within the sample chamber;

continuing flow of the CO -containing gas stream to effect an equilibration of the CO content of gas stream and blood film; and

thereafter determining the pH of the blood film.

References Cited in the file of this patent Hawk et al.: Practical Physiological Chemistry, 13th edition, McGraw-Hi1l, pages 704, 709-717.

Kauko: New Points of View in Analysis of Water, Chemical Abstracts, vol. 45, column 6538, Condensed from Ing. QVIM. (9, -55), 1950. 

1. IN A BLOOD PCO2 DETERMINATION EMPLOYING PH MEASUREMENTS OF A BLOOD SPECIMEN, THE IMPROVEMENT COMPRISING: SUPPLYING THE BLOOD SPECIMEN TO A SAMPLE CHAMBER AND THERE DETERMINING THE PH OF SAID BLOOD SPECIMEN; FORMING A FILM OF SAID BLOOD SPECIMEN WITHIN THE SAMPLE CHAMBER AND EQUILIBRATING THE BLOOD FILM WITH CO2CONTAINING GAS STREAM OF KNOWN CO2 CONTENT; AND THEREAFTER DETERMINING THE PH OF THE EQUILIBRATED BLOOD FILM. 